Soft tissue processing

ABSTRACT

The present invention is a process for preparing soft tissue such as tendons, ligaments, cartilage, fascia, dermis, human valves and human veins for implant in a human and removes cellular components and forms an decellular matrix having as major components collagens and elastins while sterilizing the tissue. The process comprises the following steps: (1) isolating from a suitable donor a desired soft tissue sample of the biological material; (2) processing and decellularizing the soft tissue including inspection for visual defects, trimming and soaking the tissue in a detergent depending on whether the tissue is fascia or dermis and rinsing same with sterile water; (3) sterilizing the soft tissue in a vacuum and soaking the tissue in an antibiotic composition or peracetic acid depending on whether the soft tissue is fascia or dermis and rinsing same; (4) processing the tissue by cutting the tissue to size and perforating the tissue; and (5) dipping the tissue in 70% ethanol and packaging the tissue.

RELATED APPLICATIONS

The present application claims priority from U.S. Provisional PatentApplication No. 60/662,078, filed Mar. 16, 2005.

FIELD OF INVENTION

The present invention is generally directed toward methods of treatmentof allograft soft tissue including decellularizing and sterilization forimplantation into another human being.

BACKGROUND OF THE INVENTION

Techniques for restoring structure and function to damaged tissue areused routinely in the area of reconstructive surgery. Tissuetransplantation is another way of restoring function by replacing orrebuilding the damaged tissue. However, problems exist when there is atransfer of biological material from one individual to another. Tissuerejection is a significant risk associated with transplantation, evenwith a good histocompatability match. Immunosuppressive drugs such ascyclosporin and FK506 are usually given to the patient to preventrejection. These immunosuppressive drugs however, have a narrowtherapeutic window between adequate immunosuppression and toxicity.Prolonged immunosuppression can weaken the immune system, which can leadto a threat of infection.

The present invention is directed toward a process for use in thepreparation of acellular, i.e. (essentially lacking in living cellsand/or non-living cells,) soft-tissue implants, in commercializablequantities. These implants are derived from tissue products derived fromanimal or human donors that contain or are devoid of cells. Thedecellularized grafts produced are significantly improved in long-termdurability and function when used in clinical applications.

Various methods have been used in the treatment of soft tissue and suchrepresentative methods are disclosed in a number of prior artapplications.

The advantages of retaining an acellular matrix, composed primarily of acollagenous component intact, has been explored by Klaus. and Duhamel(WO 84/0488)) for the production of sterile body implants. In thismethod, a variety of tissues were extracted sequentially with non-ionicand ionic detergents to yield structures essentially free of cellularmembranes, nucleic acids, lipids and cytoplasmic components. Thetreatment consists of sequential extractions with a non-denaturingdetergent and a denaturing detergent to form an acellular matrix ofcollagen.

U.S. Pat. No. 4,776,853 issued Oct. 11, 1988 is directed toward aprocess for preparing biological material for implant in a mammal'scardiovascular system, respiratory system or soft tissue. The processcomprises: (1) isolating a desired tissue sample of the biologicalmaterial from a donor; (2) extracting the tissue sample with anhypotonic buffer solution at a mild alkaline pH, the buffer solutionincluding active amounts of proteolytic inhibitors and antibiotics; (3)extracting the tissue sample with a buffered solution having a highconcentration of salt, the solution being at a mild alkaline pH andincluding a non-ionic detergent with protease inhibitors andantibiotics; (4) subjecting tissue sample to enzymatic digestion in abuffered saline solution, the enzymes consisting of purifiedprotease-free dioxyribonuclease and ribonuclease; (5) extracting thetissue sample with an anionic detergent at a mild alkaline pH; and (6)storing the tissue sample in physiologic buffered solutions.

Another soft tissue process is shown in U.S. Pat. No. 6,734,018 issuedMay 11, 2004 which is directed toward a process for preparing anacellular soft tissue graft for implantation into a mammalian system.The process extracts a soft tissue sample with an extracting solutionincluding one or more nonionic detergents and one or more endonucleases,to produce extracted tissue and treats the extracted tissue with atreating solution including one or more anionic detergents, to produce atreated tissue. The treated tissue is washed with a decontaminatingsolution including one or more decontaminating agents to produce theacellular soft tissue graft; and acellular soft tissue graft is thenstored in a storage solution comprising one or more decontaminatingagents.

The soft tissue process of the '018 patent includes the steps of:isolating from a suitable donor a desired tissue sample of thebiological material; extracting the tissue with mildly alkalinehypotonic buffered solution of an endonuclease such as Benzonase RTM anda nonionic detergent formulation such as Allowash Solution™ optionallytreating the tissue with a hypertonic buffered salt solution; extractingand treating the tissue with a mildly alkaline hypotonic bufferedsolution of sodium dodecylsulfate, optionally with 0.1 to 0.5 M sodiumchloride rendering the solution hypertonic; washing the tissue withultrapure water followed by a water solution of chlorine dioxide; andstorage in a sealed container in isotonic saline, chlorine dioxide or70% isopropanol.

It can thus be seen that the prior art processes require extensivechemical treatment with a multitude of process steps in an attempt toobtain an acellular soft tissue specimen which has limited shelf life.

SUMMARY OF THE INVENTION

The present invention is a process for preparing soft tissue for implantin a human and removes cellular components forming a decellular matrixhaving as major components; collagens and elastins while sterilizing thetissue. The process comprises the following steps:

(1) isolating from a suitable donor a desired soft tissue sample of thebiological material;

(2) processing and decellularizing the soft tissue including inspectionfor visual defects, trimming and soaking the tissue in a detergentdepending on whether the tissue is fascia or dermis and rinsing samewith sterile water;

(3) sterilizing the soft tissue by soaking the tissue in an antibioticcomposition and/or peracetic acid depending on the specific tissue andrinsing same to remove residual process chemicals;

(4) processing the tissue by cutting the tissue to size and perforatingthe tissue; and

(5) dipping the tissue in 70% ethanol and 30% water and packaging thetissue.

It is thus an object of the invention to provide decellularizedallograft soft tissue for implantation into a human being.

It is another object of the invention to provide decellularizedsterilized allograft soft tissue which is packaged for usage as animplant by a surgeon.

It is still another object of the invention to provide decellularizedsterilized allograft soft tissue which can be stored for long periods oftime for later use by a surgeon for implantation into a human being.

It is yet another object of the invention to provide decellularizedsterilized allograft soft tissue which is flexible immediately uponremoval from the package.

These and other objects, advantages, and novel features of the presentinvention will become apparent when considered with the teachingscontained in the detailed disclosure along with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow chart showing the soft tissue process;

FIG. 2 is a schematic flow chart showing the soft tissue process forfascia;

FIG. 3 is a schematic flow chart showing the soft tissue process fordermis; and

FIG. 4 is a top plan view of a soft tissue strip produced by theprocess.

DESCRIPTION OF THE INVENTION

The present invention is directed towards the preparation of allograftsoft tissue such as thawed tissue which is processed and decellularized.The preferred mode and best embodiment of the invention is shown inFIGS. 1-3.

The process uses allograft human soft tissue which has been previouslytaken from a human donor and frozen for later use. The soft tissue whichis envisioned as being used is facia, dermis, cartilage, pericardium,human valves and veins, tendons and ligaments. The soft tissue which hasbeen previously obtained from a donor and frozen is taken from thefreezer and thawed. Prior to processing, tissue is inspected for damage(holes or tears) and distinctive features (moles, warts, tattoos) whichare removed using a scalpel. Tissue is inspected for hair and the sameis removed using forceps. A visual inspection is performed to ensure thetissue has uniform thickness. Any region of non-uniformity or visiblylow thickness is removed. Thickness is recorded using a thickness gauge.To identify the orientation (dermal or epidermal side) of tissue such asskin, the skin is positioned such that the epidermis faces the processorand an incision is cut into the upper left corner of each piece oftissue to indicate the epidermal side.

After thawing it is processed and decellularized. For tendons,ligaments, muscle, fascia, muscle, pericardium and dermis soft tissue,the tissue form is inspected for visual defects and then trimmed forprocession.

When skin is used the epidermal layer is removed and the tissue isdecellularized using a 1M Sodium Chloride (NaCl) solution in a tissueflask and agitated at a speed of 65 RPM on an orbital shaker for aminimum of 12 hours, up to a maximum of 48 hours. The epidermal layer ofthe skin is removed at this time and rinsed with sterile water. Theremaining dermis is replaced in the tissue flasks filled with sterilewater and agitated on the orbital shaker for 15 minutes. The sterilewater is refreshed and the rinse procedure is repeated one more time fora total of two rinses. Once the rinse is complete, the water is replacedwith 0.1% Triton X-100 solution and agitated on the orbital shaker for aminimum of 24 hours, up to a maximum of 48 hours. The dermis is thenrinsed with sterile water, replaced in the tissue flasks filled withsterile water, and agitated on the orbital shaker at 65 RPM for 15minutes. The sterile water is refreshed and the rinse procedure isrepeated a minimum of 5 more times. A residual detergent test isperformed on the rinsate to ensure the detergent has been adequatelyremoved. The two steps utilizing 1M NaCl and 0.1% Triton soaks may becombined.

The decellularized dermis is subjected to sterilization in a solutioncontaining peracetic acid, alcohol, propylene glycol, and water andsoaked and agitated at 65 RPM under vacuum for a minimum of 4 hours, upto a maximum of 8 hours. The dermis undergoes a rinse series followedwith agitation at 65 RPM under vacuum; two 5-minute rinses, followed bytwo 10-minute rinses, followed by two 15-minute rinses for a total of 6rinses. After the last rinse, the residual test is performed on therinsate to ensure that the peracetic acid has been adequately removed.

Other tissue such as fascia, cartilage, pericardium, tendons andligaments is soaked in antibiotic soak for 1.5 to 24 hours and rinsedwith sterile phosphate buffered saline. If desired the other tissue canalso be soaked in 1M NaCl for 1.5 to 48 hours, rinsed with sterile watera plurality of times, then soaked in 0.1% Triton X-100 for 4 to 48 hrsand rinsed with sterile water until a minimal residual amount ofdetergent is reached. The tissue is soaked in peracetic acid in vacuumfor 4 hours and rinsed with sterile water from 6 to 9 times until aminimum residual amount of acid is tested.

The sterilized tissue is cut to finished size. The fascia and dermis canbe perforated with holes about 1.2 mm in diameter spaced from each other2 to 3 mm. The tissue is dipped in 70% ethanol and 30% water andpackaged.

EXAMPLE 1 Treatment of Fascia

A desired frozen soft tissue sample is isolated from a suitable donorand then thawed.

The thawed tissue is processed and decellularized and is inspected forvisual defects and trimmed.

The trimmed tissue sample is sterilized while soaking the tissue in anantibiotic soak for 1.5 to 24 hours and is rinsed with phosphatebuffered saline. If desired at the time of sterilization one or more ofthe following protease inhibitors may be added;Aminoethylbenzenesulfonyl fluoride HCL (Serine Proteases), Aprotinin(broad spectrum, serine proteases), Protease Inhibitor E-64 (CysteineProteases), Leupeptin, Hemisulfate Cysteine Proteases and trypsin-likeproteases, Pepstatin A (Aspartic Proteases) and Marmistat (MMP2). Ifdesired a solution with pH of 8.0 can be added which will inhibitlysozomal enzymes

The processed soft tissue is placed into a stainless steel containerwhich is filled with an antibiotic solution. The antibiotic solution isa packet of pre-measured antibiotics; Primaxin 0.2168 g/P_(prim),Amphotericin B 0.0297 g/P_(amph), Gentamicin 0.0369 g/P_(gent),respectively, with a tolerance of ±2.5%, and Phosphate Buffered Saline.It should be noted that one of the antibiotics is light sensitive andmust be protected from light source. The container or a plurality ofcontainers is placed on a incubator orbital shaker. The purpose of theincubator orbital shaker is to agitate the tissue in the antibioticsolution. The incubator orbital shaker is set at 120 rpm and thetemperature reading on the incubator shaker is kept in a range between35° C. to 39° C. The tissue is soaked and agitated on the incubatorshaker for a minimum of 1.5 hours and a maximum of 24 hours in theantibiotic solution.

When rinsing, the tissue is transferred to a 1000 ml polypropylene orNalgene container. In a graduated beaker 2 packets of phosphate bufferedsaline are added into 2000 ml of USP purified water and stirred untildissolved. Each polypropylene or Nalgene container containing tissue isfilled with the phosphate buffer saline solution and the cap tightened.The tissue is agitated for a minimum of 5 minutes (to a maximum of 20minutes) and the phosphate buffered saline solution is then emptied outof the container. The rinse steps are repeated; (2^(nd) rinse minimum 5minute, maximum 20 minutes phosphate buffered saline rinse). A finaladditional distilled water rinse is then completed. Once the cycle iscomplete the containers are removed from the orbital shaker. Thesolution is emptied and the processed tissue is placed on sterile wipes.The soft tissue is now ready to be measured for finishing cutting,perforating and packaging.

The treated fascia tissue is cut to size and may be frozen orlyophilized. If desired, the tissue may be perforated with theperforations 10 spaced 2-3 mm apart and each perforation preferablyhaving a diameter of about 1.2 mm. A view of the finished cut tissue isseen in FIG. 4.

The fascia tissue is immersed in 70% ethanol and 30% water and packagedin a sealed container.

EXAMPLE 2 Treatment of Dermis

Frozen donor tissue is then thawed and then rinsed to maintain moisture.The thawed tissue is processed and decellularized. If desired at thetime of decellularization one or more of the following proteaseinhibitors may be added; Aminoethylbenzenesulfonyl fluoride HCL (serineproteases) (25-100 μm, Aprotinin (broad spectrum, serine proteases)(7.5-30 μm), Protease Inhibitor E-64 (cysteine proteases) (0.05-0.0.20μm), Leupeptin, Hemisulfate (cysteine proteases) (0.05-0.0.20 μm), EDTA,Disodium (0.025-0.0.10 μm), and trypsin-like proteases, Pepstatin A(Aspartic Proteases). Marmistat (MMP2). The thawed tissue is processedand decellularized and is inspected for visual defects and trimmed.

Once all blood and lipids are removed from the skin, the water ischanged with clean sterile water. Impurities are removed from each pieceof skin with a scalpel (epidermal side up during this process). Placeeach skin piece with the epidermal side up on the cutting board or flatsurface, check the skin for damage (holes and initial tearing) and fordistinctive features (mole, warts, tattoos) and cut these impurities offusing a scalpel.

Each piece is checked for hairs and the hairs are removed with forcepsafter which the skin is rinsed with water. The skin is positioned withthe dermis side up (epidermis down) on the cutting board and rectangularskin pieces are cut by removing the rough edges of each piece with oneor more uninterrupted cuts using a scalpel and ruler. An incision is cutinto the left hand corner of each piece of skin indicating the epidermalside of the skin. A visual inspection is performed to make sure thetissue has a uniform thickness throughout the piece and regions with avisibly low or non-uniform thickness are removed. A thicknessmeasurement is then performed using a thickness gauge. An incision iscut into the upper left corner of each piece of skin so that theepidermal side of the skin is facing the processor to ensure that tissuehas a uniform thickness. The skin is decellularized in a sterile tissueculture bottle filled with IL of 1M NaCl. The bottle is sealed in aself-seal pouch and then placed the bottle on its flat side on theshaker with a set speed of 65 rpm's. The bottle(s) is checked after 12hours to see if the epidermal layers have sloughed off. After the first12 hour check, the bottle is checked every 2 hours until all epidermallayers have been sloughed. The bottles are removed from the shaker andthe NaCl is emptied from the bottle(s). The skin is removed from thebottle and placed on the cutting board with the epidermal side up. Theepidermal layers are peeled off with forceps and discarded leaving onlythe dermal layer (dermis). The bottles are rinsed with sterile water andthe peeled skin pieces (dermis) are placed back into the bottle. Thebottles are then filled with enough sterile water to submerge the tissuewhile the bottle is lying flat and the bottle is placed on the shakerwhich has a preset speed of 65 rpm's. The shaker is set to run for 15minutes. After running 15 minutes, the bottle(s) are removed and thewater is changed with clean sterile water. This rinse is repeated onemore time for a total of two times. The bottle(s),are removed from theshaker, emptied and filled with 1 L of 0.1% Triton X-100. The bottlecontaining the dermis is seated in a self-seal pouch and placed on theshaker set to the speed to 65 rpm's and allowed to shake for 24 to 48hours. The shaker is stopped after 24 hours or a later time period, thedermis is removed from the bottles and place submerged in a containerwith sterile water to rinse off the Triton X-100. The tissue is againrinsed with a sterile water for 15 minutes at 65 rpm's for irrigation torinse off the Triton X-100. The rinse is repeated 5 more times for atotal of 6 times. After rinsing a residual detergent test is performedto make sure that the detergent has been removed from the tissue.

The dermis is placed on a screen sterilized in peracetic acid for atleast 4 hours in a canister. The dermis can be soaked in peracetic acidfor 4 to 8 hours The canister stays on the shaker during the soak withthe shaker set at 65 rpm's. The dermis is initially rinsed in sterilewater on the shaker at 65 rpm=s for 5 minutes and then rinsed 5 moretimes; 2^(nd) rinse for 5 minutes, 3^(rd) and 4^(th) rinse for 10minutes and 5^(th) and 6^(th) rinse for 15 minutes. After the 6th rinse,a test is performed for the presence of the peracetic acid.

The strips of dermis are taken out of the canister using forceps andplaced into a stainless steel basin. The basin is filled with water forirrigation and the residual detergent is rinsed from the surface of theskin. A wipe is placed on the top of a cutting board and moistened withsterile water. The skin is taken from the basin and laid on the cuttingboard epidermal side down (smooth side up) and measured.

If the dermis is to be lyophilized the skin is placed in a double Tyvek®pouch and the tissue placed in a freezer at −70° on the lyophilizationstaging shelf until the lyophilization is available.

After the 6^(th) rinse or upon later removal from the lyophilization ,the dermis tissue is cut to size and perforated with the perforations 10spaced 2-3 mm apart as shown in FIG. 4 with each perforation preferablyhaving a diameter of about 1.2 mm.

The tissue may be lyophilized or is immersed in 70% ethanol and 30%water and packaged for storage in sterile foil.

The dipped tissue is laid flat on screens and placed in double Tyvek®pouches for lyophilization and 1 each Tyvek® pouch is sealed. Thepackage is stored flat in the freezer to prevent the tissue frombecoming wrinkled or deformed.

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.However, the invention should not be construed as limited to theparticular embodiments which have been described above. Instead, theembodiments described here should be regarded as illustrative ratherthan restrictive. Variations and changes may be made by others withoutdeparting from the scope of the present invention as defined by thefollowing claims:

1. A method for the treatment of soft tissue to prepare the same forimplantation into a human comprising the steps of: (a) thawing frozendonor tissue and decellularizing the donor tissue; (b) inspecting thedonor tissue for defects (c) trimming the donor tissue for processing(d) sterilizing the trimmed donor tissue with a soak in peracetic acidin a vacuum, (e) rinsing the material contained in the soak from thetissue; (f) cutting the treated tissue to a specific size; (g) immersingthe cut tissue in ethanol; and (h) packaging the ethanol soaked tissuein a sealed package.
 2. The method as claimed in claim 1 wherein saidsoft tissue is fascia.
 3. The method as claimed in claim 1 wherein saidsoft tissue is dermis.
 4. The method as claimed in claim 1 wherein saidsoft tissue is at least one tendon.
 5. The method as claimed in claim 1wherein said soft tissue is at least one ligament.
 6. The method asclaimed in claim 1 wherein said soft tissue is muscle.
 7. The method asclaimed in claim 1 wherein said soft tissue is pericardium.
 8. Themethod as claimed in claim 1 wherein said soft tissue is a human valve.9. The method as claimed in claim 1 wherein said soft tissue is a humanvein.
 10. The method as claimed in claim 1 wherein the soft tissue isdermis and after step (f), the cut tissue is perforated with a pluralityof spaced apertures.
 11. The method as claimed in claim 1 wherein afterstep (a), decellularizing adding one or more of a group of the proteaseinhibitors consisting of one or more of the following proteaseinhibitors may be added; Aminoethylbenzenesulfonyl fluoride HCL (SerineProteases), Aprotinin (broad spectrum, serine proteases), ProteaseInhibitor E-64 (Cysteine Proteases), Leupeptin, Hemisulfate CysteineProteases and trypsin-like proteases, Pepstatin A (Aspartic Proteases).Marmistat (MMP2).
 12. A method for the treatment of soft fascia tissueto prepare the same for implantation into a human comprising the stepsof: (a) thawing frozen donor fascia tissue; (b) inspecting the donortissue for defects; (c) trimming the donor tissue for processing; (d)soaking the trimmed donor tissue in a compound antibiotic mixture from1.5 to 24 hours; (e) rinsing the antibiotic mixture from the tissue withsterile water; (f) cutting the treated tissue to a specific size; (g)perforating the cut tissue with a plurality of spaced apertures; (h)immersing the perforated cut tissue in ethanol; and (i) packaging theethanol soaked perforated tissue in a sealed package.
 13. The method asclaimed in claim 12 wherein said antibiotic mixture comprises Primaxin0.2168 g/P_(prim), Amphotericin B 0.0297 g/P_(amph) and Gentamicin. 14.The method as claimed in claim 12 wherein said antibiotic mixture istaken from a group of antibiotics consisting of one or more of Primaxin,Amphotericin, and Gentamicin.
 15. The method as claimed in claim 12wherein after step (c) trimming, there is an additional step of addingone or more of a group of the following protease inhibitors consistingof Aminoethylbenzenesulfonyl fluoride HCL (Serine Proteases), Aprotinin(broad spectrum, serine proteases), Protease Inhibitor E-64 (CysteineProteases), Leupeptin, Hemisulfate Cysteine Proteases and trypsin-likeproteases, Pepstatin A (Aspartic Proteases) and Marmistat (MMP2). 16.The method as claimed in claim 12 wherein after step (d), there is anadditional step of soaking the donor tissue with phosphate bufferedsaline.
 17. A method for the treatment of soft skin tissue to preparethe same for implantation into a human comprising the steps of: (a)thawing the frozen donor tissue (b) inspecting the donor tissue fordefects (c) trimming the donor tissue for processing (d) removing theepidermal layer from the skin leaving the dermis; (e) soaking theremaining skin in 1M NaCl from 1.5 to 48 hours; (f) rinse the NaClsoaked skin with sterile water a plurality of times to remove the NaCL;(g) soak the treated skin in 0.1% Triton X-100 detergent from 1.5 to 48hours; (h) rinse the Triton X-100 detergent soaked skin with sterilewater to remove the detergent to an acceptable level; (i) sterilize theskin in a sterilization solution for 4 to 8 hours; (j) rinse the skinwith sterile water a plurality of times to reduce the acid content to apredetermined residual level; (k) cutting the treated tissue to aspecific size; (l) perforating the cut tissue with a plurality of spacedapertures; (m) immersing the perforated cut tissue in ethanol; and (n)packaging the ethanol soaked perforated tissue in a sealed package. 18.The method as claimed in claim 17 wherein after step (d), the step ofadding one or more of a group of the following protease inhibitorsconsisting of Aminoethylbenzenesulfonyl fluoride HCL (Serine Proteases),Aprotinin (broad spectrum, serine proteases), Protease Inhibitor E-64(Cysteine Proteases), Leupeptin, Hemisulfate Cysteine Proteases andtrypsin-like proteases, Pepstatin A (Aspartic Proteases) and Marmistat(MMP2).
 19. The method as claimed in claim 17 wherein the sterilizationsolution of step (e) comprises propylene glycol, ethanol 95% sterilewater and peracetic acid 35%.
 20. A method for the treatment ofcartilage to prepare the same for implantation into a human comprisingthe steps of: (a) thawing frozen cartilage tissue; (b) soaking the donortissue in a compound antibiotic mixture from 1.5 to 24 hours; (c)rinsing the antibiotic mixture from the tissue with phosphate bufferedsaline; (d) lyophilizing the treated tissue to achieve a residentialmoisture level between 0.1% to 8.0%; and (e) milling the cartilagepieces to sizes less than 1 mm.